This proposal builds upon our metabolomics expertise in untargeted and targeted metabolomics for the generation of highly sensitive and quantitative targeted multiplex assays. The idea behind generating such assays for radiation assessment and radiation injury in easily accessible biofluids (urine, blood, saliva) is to rapidly determine the extent of exposure of an individual and distinguish between the worried well and the exposed individuals that may require medical intervention. Highly quantitative approaches will be undertaken through liquid chromatography tandem mass spectrometry (LC-MS/MS) to quantify each already identified radiation biomarkers in each biofluid. Such instruments are currently used routinely in clinical laboratories, therefore maximizing the available resources for rapid evaluation of thousands of individuals during an emergency. Based on criteria for sensitivity, high signal-to-noise ratio, low signal suppression from matrix effects, and high fold changes compared to controls or relationships between pairs of metabolites, biosignatures will be assembled and concentrations calculated. The combined biosignature will be developed in a multiplex assay, effectively reducing the time between sample preparation to results. The goal is to demonstrate that this multiplex assay method has the potential to be deployed in the case of an emergency to a pre-determined network of clinical laboratories that can accept and rapidly process a high volume of samples. While the ultimate goal will be for such a panel to be predictive in all cases, even a limited false positive rate would facilitate assessment of radiation injury in a mass casualty scenario: e.g. a 1% false positive rate would reduce the number of individuals needing further evaluation by 100-fold. Additionally, this assay will be flexible as it could be enriched with biomarkers for specificity and radiation quality. This application also combines the engineering experience and capabilities of our collaborators to further develop pre-processing devices with the intention of stabilizing the sample during transport to a clinical facility. The materials to be fabricated will also aim to enrich the biosignature for the radiation-related metabolites and extract them effectively from small amounts of a biofluid (urine, serum, whole blood, saliva), transported as a stable dry membrane. Assembly of such materials in a 96 well plate will further decrease the sample preparation time and minimize human error associated with sample preparation. Our unique approach to combine LC-MS/MS applications with pre-processing materials will aim to move this technology from the feasibility stage to technology development, satisfying the needs for rapid methods for radiation injury assessment. !